This invention relates to methods for the preparation of oligomeric compounds having phosphite, phosphodiester, phosphorothioate, phosphorodithioate or other linkages, and to intermediates useful in their preparation.
Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2xe2x80x2-O-methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides and their analogs.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also xe2x80x9cDNA-protein interactions and The Polymerase Chain Reactionxe2x80x9d in Vol. 2 of Current Protocols In Molecular Biology, supra.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, Tm), to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
The chemical literature discloses numerous processes for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One of the most popular processes is the phosphoramidite technique (see, e.g., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references cited therein), wherein a nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Oxidation of the phosphite linkage followed by hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
The phosphoramidite technique, however, has significant disadvantages. For example, cyanoethyl phosphoramidite monomers are quite expensive. Although considerable quantities of monomer go unreacted in a typical phosphoramidite coupling, unreacted monomer can be recovered, if at all, only with great difficulty.
Another disadvantage of using a xcex2-eliminating cyanoethoxy group is formation of acrylonitrile upon removal of the phosphorus protecting group. Acrylonitrile is a highly toxic agent as well as a suspected carcinogen (See 1994-1995 Aldrich Chemical Company Catalog, at page 32). Acrylonitrile is also suspected of forming cyclic structures with thymidine resulting in oligomeric compounds having decreased hybridization ability. These modified oligomeric compounds are undesirable because they are difficult to separate from the desired oligomeric compound.
Consequently, there remains a need in the art for synthetic methods that will overcome these problems.
Several processes are known for the solid phase synthesis of oligonucleotide compounds. These are generally disclosed in the following U.S. Patents: U.S. Pat. No. 4,458,066; issued Jul. 3, 1984; U.S. Pat. No. 4,500,707, issued Feb. 19, 1985; and U.S. Pat. No. 5,132,418, issued Jul. 21, 1992. Additionally, a process for the preparation of oligonucleotides using phosphoramidite intermediates is disclosed in U.S. Pat. No. 4,973,679, issued Nov. 27, 1990.
A process for the preparation of phosphoramidites is disclosed in U.S. Pat. No. 4,415,732, issued Nov. 15, 1983.
Phosphoramidite nucleoside compounds are disclosed in U.S. Pat. No. 4,668,777, issued May 26, 1987.
A process for the preparation of oligonucleotides using a xcex2-eliminating phosphorus protecting group is disclosed in U.S. Pat. No. Re. 34,069, issued Sep. 15, 1992.
A process for the preparation of oligonucleotides using a xcex2-eliminating or allylic phosphorus protecting group is disclosed in U.S. Pat. No. 5,026,838, issued Jun. 25, 1991.
In one aspect of the present invention, methods are provided for the preparation of oligomeric compounds comprising a moiety having the Formula I: 
wherein:
A is a monocyclic or bicyclic aromatic ring system;
R11 and R12 are each independently H, alkyl, aryl, heteroalkyl, heteroaryl, alkaryl, or aralkyl;
or R11 and R12 together with the carbon atoms to which they are attached form an optionally substituted aliphatic or aromatic ring having from 4 to 6 ring atoms;
X4 is alkaryl, aralkyl, sulfoxyl, sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, or substituted thio, wherein said substituent is alkyl, aryl, or alkaryl;
or X4 is a group of formula xe2x80x94C (xe2x95x90O)xe2x80x94(O)aaxe2x80x94R40 where aa is 0 or 1 and R40 is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl, aryl, aralkyl or heteroaryl groups are optionally substituted with one or more alkyl, aryl, aralkyl, halo or acetyl groups;
or X4 is a group of formula xe2x80x94(xe2x80x94CH2xe2x80x94CH2xe2x80x94)dSi(R9)3 where d is 0 or 1;
each R9 is, independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6 to about 10 carbon atoms;
X1 and X5 are each independently O or S; comprising:
(a) providing a compound having the Formula II: 
wherein:
each R1, is, independently, H, hydroxyl, C1-C20 alkyl, C3-C20 alkenyl, C2-C20 alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or polyether;
or R1 is a group of formula Zxe2x80x94R22xe2x80x94(R23)v;
Z is O, S, NH, or Nxe2x80x94R22xe2x80x94(R23)v;
R22 is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl;
R23 is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides;
v is from 0 to about 10;
or R1 has the formula: 
y1 is 0 or 1;
y2 is independently 0 to 10;
y3 is 1 to 10;
E is C1-C10 alkyl, N(Q1) (Q2) or Nxe2x95x90C(Q1) (Q2);
each Q1 and Q2 is, independently, H, C1-C10 alkyl, substituted C1-C10 alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support; or Q1 and Q2, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
or R1 has one of formula XI or XII: 
xe2x80x83wherein
Z0 is O, S, or NH;
q1 is from 0 to 10;
q2 is from 1 to 10;
q3 is 0 or 1;
q4 is, 0, 1 or 2;
Z4 is OM1, SM1, or N(M1)2;
each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)M2, C(xe2x95x90O)N(H)M2 or OC(xe2x95x90O)N(H)M2;
M2 is H or C1-C8 alkyl;
Z1, Z2 and Z3 comprise a ring system having from about 4 to about 7 carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur, and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic; and
Z5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q1) (Q2), OQ1, halo, SQ1 or CN;
R3 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support;
each B, independently, is a naturally occurring or non-naturally occurring nucleobase or a protected naturally occurring or non-naturally occurring nucleobase;
n is 0 to about 50;
M is an optionally protected internucleoside linkage;
R5 is xe2x80x94N (R6)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen; and
R6 is straight or branched chain alkyl having from 1 to 10 carbons; and
(b) reacting the compound of Formula II with a compound having Formula III: 
wherein m is 0 to about 50;
R3a is hydrogen;
R2 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support, provided that R2 and R3a are not both simultaneously a linker connected to a solid support; to form the oligomeric compound.
Some preferred embodiments of the methods of the invention further comprise the step of oxidizing or sulfurizing the oligomeric compound. In some preferred embodiments, the methods of the invention further comprise transforming the oxidized or sulfurized oligomeric compound to form a further compound having the Formula III, where m is increased by 1. Other prefered embodiments further comprise a capping step, performed prior to or subsequent to oxidation or sulfurization.
In some preferred embodiments, the methods of the invention further comprising the step of cleaving the oligomeric compound from the solid support to produce a compound having the Formula IV: 
In some preferred embodiments of the methods of the invention, A is phenyl or a naphthalene.
In further preferred embodiments of the methods of the invention, X4 is benzoyl, acetyl (xe2x80x94C(xe2x95x90O)xe2x80x94CH3) or levulinyl.
In some partiularly preferred embodiments, X4 is benzoyl, acetyl or levulinyl, A is phenyl, with the moiety xe2x80x94OX4 being in the ortho or para position thereof, with the ortho position being more preferred.
In further preferred embodiments of the methods of the invention, X4 is benzoyl, acetyl or levulinyl, A is a naphthalene ring connected to X5 at the 1-position, with the moiety xe2x80x94OX4 being in the 5- or 6-position of the naphthalene ring.
In further preferred embodiments of the invention, each R6 is isopropyl.
In some especially preferred embodiments of the invention, n is 0. In further prefered embodiments, at least one of X1 and X5 is O. More preferably, X1 and X5 are each O.
In some especially preferred emboiments, n is 0; X4 is benzoyl, acetyl or levulinyl; A is phenyl; xe2x80x94OX4 is in the ortho or para position, with the ortho position being more preferred; X1 and X5 are each O; and R5 is diisopropylamino.
In some preferred embodiments, the compound of Formula II is obtained by reaction of a compound having Formula V: 
with a compound having the Formula VI: 
in the presence of an acid, preferably tetrazole. Preferably, R5 is N,N-diisopropylamino.
In other preferred embodiments, the compound of Formula II is obtained by (a) reacting a compound having Formula-V with a chlorophosphine compund of formula ClP(R5)2 in the presence of a base; and
(b) contacting the product of step (a) with a compound of Formula XX: 
in the presence of an acid. Preferably, the chlorophosphine compound has the formula ClP[(i-Pr)2N]2.
Also provided in accordance with the present invention are compounds having Formula VII: 
wherein:
A is a monocyclic or bicyclic aromatic ring system;
R11 and R12 are each independently H, alkyl, aryl, heteroaryl, alkaryl, or aralkyl;
or R11 and R12 together with the carbon atoms to which they are attached form an optionally substituted aliphatic or aromatic ring having from 4 to 6 ring atoms;
X4 is alkaryl, aralkyl, sulfoxyl, sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, or substituted thio, wherein said substituent is alkyl, aryl, or alkaryl;
or X4 is a group of formula xe2x80x94C (xe2x95x90O)xe2x80x94(O)aaxe2x80x94R40 where aa is 0 or 1 and R40 is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl, aryl, aralkyl or heteroaryl groups are optionally substituted with one or more alkyl, aryl, aralkyl, halo or acetyl groups;
or X4 is a group of formula xe2x80x94(xe2x80x94CH2xe2x80x94CH2xe2x80x94)dSi(R9)3 where d is 0 or 1;
each R9 is, independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6 to about 10 carbon atoms;
X1 and X5 are each independently O or S;
D is (R7) (R8) Pxe2x80x94or (R7) (R8) P (xe2x95x90X2)xe2x80x94;
R8 is R5, or has the Formula VIII: 
wherein:
each R1, is, independently, H, hydroxyl, C1-C20 alkyl, C3-C20 alkenyl, C2-C20 alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or polyether;
or R1 is a group of formula Zxe2x80x94R22xe2x80x94(R23)v;
Z is O, S, NH, or Nxe2x80x94R22xe2x80x94(R23)v;
R22 is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl;
R23 is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides;
v is from 0 to about 10;
or R1 has the formula: 
y1 is 0 or 1;
y2 is independently 0 to 10;
y3 is 1 to 10;
E is C1-C10 alkyl, N(Q1) (Q2) or Nxe2x95x90C(Q1) (Q2);
each Q1 and Q2 is, independently, H, C1-C10 alkyl, substituted C1-C10 alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support; or Q1 and Q2, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
or R1 has one of formula XI or XII: 
xe2x80x83wherein
Z0 is O, S, or NH;
q1 is from 0 to 10;
q2 is from 1 to 10;
q3 is 0 or 1;
q4 is, 0, 1 or 2;
Z4 is OM1, SM1, or N(M1) 2;
each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)M2, C(xe2x95x90O)N(H)M2 or OC(xe2x95x90O)N(H)M2;
M2 is H or C1-C8 alkyl;
Z1, Z2 and Z3 comprise a ring system having from about 4 to about 7 carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur, and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic; and
Z5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q1) (Q2), OQ1, halo, SQ1 or CN;
each X2 is O or S;
R5 is xe2x80x94N(R6)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
M is an optionally protected internucleoside linkage;
m is 0 to about 50;
each B, independently, is a naturally occurring or non-naturally occurring nucleobase or a protected naturally occurring or non-naturally occurring nucleobase; and
R7 is R5, or has the Formula IX: 
wherein:
R3 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support;
n is 0 to about 50; with the proviso that the sum of m and n do not exceed 50.
In some preferred embodiments, X4 is benzoyl, acetyl or levulinyl, with acetyl being preferred.
In some partiularly preferred embodiments, X4 is benzoyl, acetyl or levulinyl, A is phenyl, with the moiety xe2x80x94OX4 being in the ortho or para position, with the ortho position being more preferred, and R11 and R12 are each H.
In further preferred embodiments of the methods of the invention, X4 is benzoyl, acetyl or levulinyl, A is a naphthalene ring connected to X5 at the 1-position, with the moiety xe2x80x94OX4 being at the 5- or 6-position of the naphthalene ring, and R11 and R12 are each H.
In some preferred embodiments, at least one of X1 and X5 is O. In more preferred embodiments, X1 and X5 are each O.
In particularly preferred embodiments, X4 is benzoyl, acetyl or levulinyl, A is phenyl with xe2x80x94OX4 being in the ortho or para position, X1 and X5 are each O, and R11 and R12 are each H.
In some preferred embodiments, R8 is R5. In further preferred embodiments, n is 0. In still further preferred embodiments, R8 is R5, n is 0, X4 is benzoyl, acetyl or levulinyl, A is phenyl with xe2x80x94OX4 attached at the ortho or para position; X1 and X5 are each O, and R11 and R12 are each H.
In some preferred embodiments, R8 has the Formula VIII, and R7 has the Formula IX. In further preferred embodiments, R8 has the Formula VIII, and R7 has the Formula IX, and n is 0. In still further preferred embodiments, R8 has the Formula VIII, and R7 has the Formula IX, and n is 0 and m is 0. In still further preferred embodiments, R8 has the Formula VIII, and R7 has the Formula IX, n is 0, X4 is benzoyl, acetyl or levulinyl, A is phenyl with xe2x80x94OX4 attached at the ortho or para position, X1 and X5 are each O, R5 is diisopropylamino, and R11 and R12 are each H.
In some preferred embodiments, at least one of X1 and X5 is S. In further preferred embodiments, A is (R7) (R8)Pxe2x80x94.
In some preferred embodiments, the present invention provides compounds comprising a moiety of Formula: 
wherein the constituent variables are as previously defined.
Preferably, the moiety A is phenyl with xe2x80x94OX4 attached at the ortho or para position, with the ortho position being preferred; or A is naphthalene connected to X5 at the 1-position, and the moiety xe2x80x94OX4 is attached to the 5- or 6-position of the naphthalene ring. In especially preferred embodiments, X4 is benzoyl, acetyl or levulinyl, A is phenyl with xe2x80x94OX4 is in the ortho or para position, and X1 and X5 are each O.
The present invention also provides compounds having Formula X: 
or Formula XI: 
wherein m and n are each independently from 0 to about 50, provided that the sum of m and n does not exceed 50; and the other constituent variables are as previously defined.
In some preferred embodiments, R2 is a linker connected to a solid support.
Also provided in accordance with the present invention are methods for the preparation of a compound of Formula II: 
wherein the consitituent variables are as previously defined, comprising:
(a) selecting a 5xe2x80x2-protected nucleoside having Formula V: 
and
(b) reacting the nucleoside with a compound having the Formula VI: 
in the presence of an acid.
The present invention also provides methods for the preparation of a compound of Formula II comprising:
(a) selecting a 5xe2x80x2-protected nucleoside of Formula V: 
(b) reacting the protected nucleoside with a chlorophosphine compund of formula ClP(R5)2 in the presence of a base; and
(c) contacting the product of step (b) with a compound of Formula XX: 
in the presence of an acid;
to form the nucleoside phosphoramidite.
In some preferred embodiments of the compounds of Formulas X and XI m and n are each 0.
Also provided in accordance with the present invention are compounds having the formula: 
wherein A, X1, X4 X5, R11 and R12 are as defined above, X2 is halogen, and X3 is xe2x80x94N(R6)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen. In further preferred embodiments, A is phenyl with xe2x80x94Oxe2x80x94X4 in the ortho or para position, X1 and X5 are O, and R11 and R12 are each H, and X4 is benzoyl, acetyl or levulinyl. In still further preferred embodiments, X3 is xe2x80x94N(R6) 2 where R6 is isopropyl. Preferably, X2 is chlorine.
The present invention also provides products produced by the methods of the invention.
The present invention provides methods for the preparation of oligomeric compounds having phosphite, phosphodiester, phosphorothioate, or phosphorodithioate linkages, and to intermediates useful in their preparation.
In some preferred embodiments of the invention, methods are provided for the preparation of an oligomeric compound comprising at least one moiety having the Formula I: 
wherein:
A is a monocyclic or bicyclic aromatic ring system;
R11 and R12 are each independently H, alkyl, aryl, heteroaryl, alkaryl, or aralkyl;
or R11 and R12 together with the carbon atoms to which they are attached form an optionally substituted aliphatic or aromatic ring having from 4 to 6 ring atoms;
X4 is alkaryl, aralkyl, sulfoxyl, sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, or substituted thio, wherein said substituent is alkyl, aryl, or alkaryl;
or X4 is a group of formula xe2x80x94C(xe2x95x90O)xe2x80x94(O)aaxe2x80x94R40 where aa is 0 or 1 and R40 is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl, aryl, aralkyl or heteroaryl groups are optionally substituted with one or more alkyl, aryl, aralkyl, halo or acetyl groups;
or X4 is a group of formula xe2x80x94(xe2x80x94CH2xe2x80x94CH2xe2x80x94)dSi(R9)3 where d is 0 or 1;
each R9 is, independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6 to about 10 carbon atoms;
X1 and X5 are each independently O or S; comprising:
(a) providing a compound having the Formula II: 
wherein:
each R1, is, independently, H, hydroxyl, C1-C20 alkyl, C3-C20 alkenyl, C2-C20 alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or polyether;
or R1 is a group of formula Zxe2x80x94R22xe2x80x94(R23)v;
Z is O, S, NH, or Nxe2x80x94R22xe2x80x94(R23)v;
R22 is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl;
R23 is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, O-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmaco-dynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides;
v is from 0 to about 10;
or R1 has the formula: 
y1 is 0 or 1;
y2 is independently 0 to 10;
y3 is 1 to 10;
E is C1-C10 alkyl, N(Q1) (Q2) or Nxe2x95x90C(Q1) (Q2);
each Q1 and Q2 is, independently, H, C1-C10 alkyl, substituted C1-C10 alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support; or Q1 and Q2, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
or R1 has one of formula XI or XIII: 
xe2x80x83wherein
Z0 is O, S, or NH;
q1 is from 0 to 10;
q2 is from 1 to 10;
q3 is 0 or 1;
q4 is, 0, 1 or 2;
Z4 is OM1, SM1, or N(M1)2;
each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)M2, C(xe2x95x90O)N(H)M2 or OC(xe2x95x90O)N(H)M2;
M2 is H or C1-C8 alkyl;
Z1, Z2 and Z3 comprise a ring system having from about 4 to about 7 carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur, and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic; and
Z5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q1) (Q2), OQ1, halo, SQ1 or CN;
R3 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support;
each B, independently, is a naturally occurring or non-naturally occurring nucleobase or a protected naturally occurring or non-naturally occurring nucleobase;
n is 0 to about 50;
M is an optionally protected internucleoside linkage;
R5 is xe2x80x94N(R6)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen; and
R6 is straight or branched chain alkyl having from 1 to 10 carbons; and
(b) reacting the compound of Formula II with a compound having Formula III: 
xe2x80x83wherein m is 0 to about 50;
R3a is hydrogen;
R2 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support, provided that R2 and R3a are not both simultaneously a linker connected to a solid support;
to form the oligomeric compound.
The methods of the present invention are useful for the preparation of oligomeric compounds containing monomeric subunits that are joined by a variety of linkages, including phosphite, phosphodiester, phosphorothioate, and/or phosphorodithioate linkages. As used herein, the term xe2x80x9coligomeric compoundxe2x80x9d is used to refer to compounds containing a plurality of nucleoside monomer subunits that are joined by internucleoside linkages, preferably phosphorus-containing linkages, such as phosphite, phosphodiester, phosphorothioate, and/or phosphorodithioate linkages. The term xe2x80x9coigomeric compoundxe2x80x9d therefore includes naturally occurring oligonucleotides, their analogs, and synthetic oligonucleotides. Monomer or higher order synthons having Formulas II or III above include both native (i.e., naturally occurring) and synthetic (e.g., modified native or totally synthetic) nucleosides and nucleotides.
In some preferred embodiments, a phosphoramidite protected at the 5xe2x80x2-position is reacted with the 3xe2x80x2-hydroxyl group of a compound of Formula III to produce phosphite compound containing the linkage of Formula I. Preferably, capping and/or oxidation or sulfurization steps are then performed to produce a compound of Formula IV.
Methods for coupling compounds of Formula II and Formula III of the invention include both solution phase and solid phase chemistries. Representative solution phase techniques are described in U.S. Pat. No. 5,210,264, which is assigned to the assignee of the present invention. In preferred embodiments, the methods of the present invention are employed for use in iterative solid phase oligonucleotide synthetic regimes. Representative solid phase techniques are those typically employed for DNA and RNA synthesis utilizing standard phosphoramidite chemistry, (see, e.g., Protocols For Oligonucleotides And Analogs, Agrawal, S., ed., Humana Press, Totowa, N.J., 1993, hereby incorporated by reference in its entirety). A preferred synthetic solid phase synthesis utilizes phosphoramidites as activated phosphate compounds. In this technique, a phosphoramidite monomer is reacted with a free hydroxyl on the growing oligomer chain to produce an intermediate phosphite compound, which is subsequently oxidized to the pv state using standard methods. This technique is commonly used for the synthesis of several types of linkages including phosphodiester, phosphorothicate, and phosphorodithioate linkages.
Typically, the first step in such a process is attachment of a first monomer or higher order subunit containing a protected 5xe2x80x2-hydroxyl to a solid support, usually through a linker, using standard methods and procedures known in the art. See for example, Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991, hereby incorporated by reference in its entirety. The support-bound monomer or higher order first synthon is then treated to remove the 5xe2x80x2-protecting group, to form a compound of Formula III wherein R2 is a linker connected to a solid support. Typically, this is accomplished by treatment with acid. The solid support bound monomer is then reacted with a compound of Formula II to form a compound of Formula IV, which has a phosphite or thiophosphite linkage of Formula I. In preferred embodiments, synthons of Formula II and Formula III are reacted under anhydrous conditions in the presence of an activating agent such as, for example, 1H-tetrazole, 5-(4-nitrophenyl)-1H-tetrazole, or diisopropylamino tetrazolide.
In some preferred embodiments, phosphite or thiophosphite compounds containing a linkage of Formula I are oxidized or sulfurized as shown below to produce compounds having a linkage of Formula XII, where X1 and X2 can each be O or S: 
Choice of oxidizing or sulfurizing agent will determine whether the linkage of Formula I will be oxidized or sulfurized to a phosphotriester, thiophosphotriester, or a dithiophosphotriester linkage.
It is generally preferable to perform a capping step, either prior to or after oxidation or sulfurization of the phosphite triester, thiophosphite triester, or dithiophosphite triester. Such a capping step is generally known to be beneficial by preventing shortened oligomer chains, by blocking chains that have not reacted in the coupling cycle. One representative reagent used for capping is acetic anhydride. Other suitable capping reagents and methodologies can be found in U.S. Pat. No. 4,816,571, issued Mar. 28, 1989, hereby incorporated by reference in its entirety.
Treatment with an acid removes the 5xe2x80x2-hydroxyl protecting group, and thus transforms the solid support bound oligomer into a further compound of Formula III wherein R3a is hydrogen, which can then participate in the next synthetic iteration; i.e., which can then be reacted with a further compound of Formula II. This process is repeated until an oligomer of desired length is produced.
The completed oligomer is then cleaved from the solid support. The cleavage step, which can precede or follow deprotection of protected functional groups, will in prefered embodiments yield a compound having Formula IV wherein R2 is hydrogen. During cleavage, the linkages between monomeric subunits are converted from phosphotriester, thiophosphotriester, or dithiophosphotriester linkages to phosphodiester, phosphorothioate, or phosphorodithioate linkages.
Without intending that the invention be bound by any particular theory, it is believed that the loss of the oxygen or sulfur protecting group where X4 is an alkanoyl (e.g., acetyl) group occurs via a fragmentation mechanism, illustrated in Scheme I below for embodiments wherein moiety A is phenyl with the group xe2x80x94OX4 (exemplified as an acetyl group) in the para position: 
In this mechanism, a nucleophile (for example, ammonia) first attacks the carbonyl carbon of the acetoyl group. The resonant movement of electrons as depicted in Scheme I above is believed to cause the loss of the oxygen or sulfur protecting group via a fragmentation, thereby forming a phosphodiester, phosphorothioate, or phosphorodithioate linkage. The other products of the deprotection are Nuxe2x80x94C(xe2x95x90O)xe2x80x94CH3, p-quinone, and ethylene gas.
The mechanism for embodiments wherein moiety A is phenyl with the group xe2x80x94OX4 (exemplified as an acetyl group) attached to the ortho position is shown below in Scheme II: 
The products of the deprotection are the unprotected linkage, Nu-C(xe2x95x90O)xe2x80x94CH3, o-quinone, and ethylene gas.
In some preferred embodiments of the compounds of the invention, substituent X4 is selected such that it facilitates attack by a nucleophile, or a base. Accordingly, X4 can be any of a variety of substituents, provided that it does not otherwise interfere with the methods of the invention. Preferred non-silyl X4 groups include alkaryl groups, sulfoxyl groups, sulfonyl groups, thio groups, substituted sulfoxyl groups, substituted sulfonyl groups, or substituted thio groups, wherein the substituents are selected from the group consisting of alkyl, aryl, or alkaryl. More preferred non-silyl X4 groups include compounds of formula xe2x80x94C(xe2x95x90O)xe2x80x94(O)aaxe2x80x94R40 where aa is 0 or 1 and R40 is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl, aryl, aralkyl or heteroaryl groups are optionally substituted with one or more alkyl, aryl, aralkyl, halo or acetyl groups. Particularly preferred X4 groups include acetyl (xe2x80x94C(xe2x95x90O)xe2x80x94CH3), benzoyl (xe2x80x94C(xe2x95x90O)xe2x80x94Ph), phenylacetyl (xe2x80x94C(xe2x95x90O)xe2x80x94CH2xe2x80x94Ph) and levulinyl (xe2x80x94C(xe2x95x90O)xe2x80x94(CH2)2xe2x80x94C(xe2x95x90O) xe2x80x94CH3) groups.
In one embodiment of the invention the moiety xe2x80x94OX4 forms a carbonate or substituted carbonate group. In some preferred embodiments, X4 has the formula xe2x80x94C(xe2x95x90O)xe2x80x94(O)aaxe2x80x94R40 where aa is 1 and R40 is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl, aryl, aralkyl or heteroaryl groups are optionally substituted with one or more alkyl, aryl, aralkyl, halo or acetyl groups. Carbonate protecting groups are discussed in for example, Green and Wuts, Protective Groups in Organic Synthesis, 2d edition, John Wiley and Sons, New York, 1991, pages 104-105 et al., incorporated herein by reference.
X4 can also be a group of formula a group of formula xe2x80x94(xe2x80x94CH2xe2x80x94CH2xe2x80x94)dSi(R9)3 where d is 0 or 1, and each R9 is, independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6 to about 10 carbon atoms. While not wishing to be bound by a particular theory, it is believed that the loss of the oxygen or sulfur where X4 is a trisubstituted silyl moiety, occurs via a fragmentation mechanism, illustrated in Scheme III below for embodiments wherein A is phenyl with xe2x80x94OX4 at the para position: 
In this mechanism, a nucleophile attacks the silyl silicom atom, and the resonant movement of electrons as depicted in Scheme III above is believed to cause the loss of the oxygen or sulfur protecting group via a fragmentation mechanism, thereby forming a phosphodiester, phosphorothioate, or phosphorodithioate linkage. The other products of the deprotection are believed to be ethylene gas, p-quinone, and a compound of formula Nuxe2x80x94Si(R9)3. For embodiments wherein the moiety (R9)3Sixe2x80x94 is in the ortho position of the phenyl ring, the analogous fragmentation is beleived to result in the same products, except for the production of o-quinone instead of p-quinone. For embodiments wherein d is 1, it is believed that a similar fragmentation mechanism would produce the same products, and one additional mole of ethylene.
A wide variety of bases or nucleophiles can be used to initiate the fragmentation of the oxygen or sulfur protecting groups described herein. These include ammonium hydroxide, fluoride ion, alkyl amines, aqueous bases, and alkyl amines in combination with ammonium hydroxide. The resulting products include phosphate, phosphorothioate, and phosphorodithioate containing compounds.
Contact with fluoride ion preferably is effected in a solvent such as tetrahydrofuran, acetonitrile, dimethoxyethane, or water. Fluoride ion preferably is provided in the form of one or more salts selected from tetraalkylammonium fluorides (e.g., tetrabutylammonium fluoride (TBAF)), potassium fluoride, or cesium fluoride.
Preferably, conditions for removal of the oxygen or sulfur protecting group via fragmentation mechanisms described above also effect cleavage of the oligomeric compound from the solid support.
The methods of the present invention are useful for the preparation of oligomeric compounds from monomeric or oligomeric amidite synthons, for example synthons having Formula II. The internucleoside linkages of such oligomeric amidite synthons, represented by moiety M in the compounds and methods described herein, can be any internucleoside linkage as is known in the art, including phosphorus based linking groups such as phosphite, phosphodiester, phosphorothioate, and phosphorodithioate linkages, and other linkages known in the art. Such linkages can be protected, i.e., they can bear, for example, phosphate protecting groups. Included with the definition of internucleoside linkages are groups described herein, having the Formula: 
In preferred embodiments, the methods of the invention are used for the preparation of oligomeric compounds including oligonucleotides and their analogs. As used herein, the term xe2x80x9coligonuclotide analogxe2x80x9d means compounds that can contain both naturally occurring (i.e. xe2x80x9cnaturalxe2x80x9d) and non-naturally occurring (xe2x80x9csyntheticxe2x80x9d) moieties, for example, nucleosidic subunits containing modified sugar and/or nucleobase portions. Such oligonucleotide analogs are typically structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic wild type oligonucleotides. Thus, oligonucleotide analogs include all such structures which function effectively to mimic the structure and/or function of a desired RNA or DNA strand, for example, by hybridizing to a target. The term synthetic nucleoside, for the purpose of the present invention, refers to a modified nucleoside. Representative modifications include modification of a heterocyclic base portion of a nucleoside to give a non-naturally occurring nucleobase, a sugar portion of a nucleoside, or both simultaneously.
Representative nucleobases useful in the compounds and methods described herein include adenine, guanine, cytosine, uridine, and thymine, as well as other non-naturally occurring and natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine. Further naturally and non naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613-722 (see especially pages 622 and 623, and in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed., John Wiley and Sons, 1990, pages 858-859, Cook, P. D., Anti-Cancer Drug Design, 1991, 6, 585-607, each of which are hereby incorporated by reference in their entirety. The term xe2x80x98nucleosidic basexe2x80x99 is further intended to include heterocyclic compounds that can serve as like nucleosidic bases including certain xe2x80x98universal basesxe2x80x99 that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Especially mentioned as a universal base is 3-nitropyrrole.
As used herein the term xe2x80x9c2xe2x80x2-substituent groupxe2x80x9d denotes groups attached to the 2xe2x80x2 position of the ribosyl moiety, with or without an oxygen atom.
Preferred 2xe2x80x2-substituent groups described herein are represented in the compounds described herein by the variable R1, which can be independently, H, hydroxyl, C1-C20 alkyl, C3-C20 alkenyl, C2-C20 alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or polyether;
or R1 is a group of formula Zxe2x80x94R22xe2x80x94(R23)v;
Z is O, S, NH, or Nxe2x80x94R22xe2x80x94(R23)v;
R22 is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl;
R23 is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides;
v is from 0 to about 10;
or R1 has the formula: 
y1 is 0 or 1;
y2 is independently 0 to 10;
y3 is 1 to 10;
E is C1-C10 alkyl, N(Q1) (Q2) or Nxe2x95x90C(Q1) (Q2);
each Q1 and Q2 is, independently, H, C1-C10 alkyl, substituted alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support; or Q1 and Q2, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
or R1 has one of formula XI or XII: 
xe2x80x83wherein
Z0 is O, S, or NH;
q1 is from 0 to 10;
q2 is from 1 to 10;
q3 is 0 or 1;
q4 is, 0, 1 or 2;
Z4 is OM1, SM1, or N(M1)2;
each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C (xe2x95x90NH)N(H)M2, C(xe2x95x90O)N(H)M2 or OC(xe2x95x90O)N(H)M2;
M2 is H or C1-C8 alkyl;
Z1, Z2 and Z3 comprise a ring system having from about 4 to about 7 carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur, and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic; and
Z5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q1) (Q2), OQ1, halo, SQ1 or CN.
Representative 2xe2x80x2xe2x80x94Oxe2x80x94sugar substituents of formula XI are disclosed in U.S. patent application Ser. No.: 09/130,973, filed Aug. 7, 1998, entitled Capped 2xe2x80x2-Oxyethoxy Oligonucleotides, hereby incorporated by reference in its entirety.
Representative cyclic 2xe2x80x2xe2x80x94Oxe2x80x94sugar substituents of formula XII are disclosed in U.S. patent application Ser. No.: 09/123,108, filed Jul. 27, 1998, entitled RNA Targeted 2xe2x80x2-Modified Oligonucleotides that are Conformationally Preorganized, hereby incorporated by reference in its entirety.
One particularly preferred group includes 2xe2x80x2-methoxyethoxy [2xe2x80x2xe2x80x94Oxe2x80x94CH2CH2OCH3, also known as 2xe2x80x2xe2x80x94Oxe2x80x94(2-methoxyethyl) or 2xe2x80x2-MOE] (Martin et al., Helv. Chim. Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. A further referred modification includes 2xe2x80x2-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2xe2x80x2-DMAOE, as described in co-owned U.S. patent application Ser. No. 09/016,520, filed on Jan. 30, 1998, the contents of which are herein incorporated by reference. Other preferred modifications include 2xe2x80x2-methoxy (2xe2x80x2xe2x80x94Oxe2x80x94CH3) and 2xe2x80x2-aminopropoxy (2xe2x80x2xe2x80x94OCH2CH2CH2NH2).
Further preferred 2xe2x80x2-sugar modifications amenable to the present invention include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, and polyethers of the formula (O-alkyl)m where m is 1 to about 10. Preferred among these polyethers are linear and cyclic polyethylene glycols (PEGs), and (PEG)-containing groups, such as crown ethers and those which are disclosed by Ouchi, et al., Drug Design and Discovery 1992, 9, 93, Ravasio, et al., J. Org. Chem. 1991, 56, 4329, and Delgardo et. al., Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249, each of which are hereby incorporated by reference in their entirety. Further sugar modifications are disclosed in Cook, P. D., Anti-Cancer Drug Design, 1991, 6, 585-607. Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl amino substitution is described in U.S. patent application Ser. No. 08/398,901, filed Mar. 6, 1995, entitled Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2xe2x80x2 and 5xe2x80x2 Substitutions, hereby incorporated by reference in its entirety.
Additional 2xe2x80x2 sugar modifications amenable to the present invention include 2xe2x80x2-SR and 2xe2x80x2-NR2 groups, where each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl. 2xe2x80x2-SR nucleosides are disclosed in U.S. Pat. No. 5,670,633, issued Sep. 23, 1997, hereby incorporated by reference in its entirety. The incorporation of 2xe2x80x2-SR monomer synthons are disclosed by Hamm et al., J. Org. Chem., 1997, 62, 3415-3420. 2xe2x80x2-NR2 nucleosides are disclosed by Goettingen, M., J. Org. Chem., 1996, 61, 6273-6281; and Polushin et al., Tetrahedron Lett., 1996, 37, 3227-3230.
Sugars having O-substitutions on the ribosyl ring are also amenable to the present invention. Representative substitutions for ring O include S, CH2, CHF, and CF2, see, e.g., Secrist, et al., Abstract 21, Program and Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides and their Biological Applications, Park City, Utah, September 16-20, 1992, hereby incorporated by reference in its entirety. Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3xe2x80x2 position of the sugar on the 3xe2x80x2 terminal nucleotide and the 5xe2x80x2 position of 5xe2x80x2 terminal nucleotide. For example, one additional modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides and Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).
As used herein, the term xe2x80x9calkylxe2x80x9d includes but is not limited to straight chain, branch chain, and alicyclic hydrocarbon groups. Alkyl groups of the present invention may be substituted. Representative alkyl substituents are disclosed in U.S. Pat. No. 5,212,295, at column 12, lines 41-50, hereby incorporated by reference in its entirety. As used herein, the term xe2x80x9clower alkylxe2x80x9d is intended to mean alkyl having 6 or fewer carbons.
As used herein, the term xe2x80x9cara_kylxe2x80x9d denotes alkyl groups which bear aryl groups, for example, benzyl groups. The term xe2x80x9calkarylxe2x80x9d denotes aryl groups which bear alkyl groups, for example, methylphenyl groups. As used herein the term xe2x80x9carylxe2x80x9d denotes aromatic cyclic groups including but not limited to phenyl, naphthyl, anthracyl, phenanthryl, and pyrenyl.
As used herein, the term xe2x80x9calkanoylxe2x80x9d has its accustomed meaning as a group of formula xe2x80x94C(xe2x95x90O)-alkyl. A preferred alkanoyl group is the acetyl group.
In general, the term xe2x80x9cheteroxe2x80x9d denotes an atom other than carbon, preferably but not exclusively N, O, or S. Accordingly, the term xe2x80x9cheterocycloalkylxe2x80x9d denotes an alkyl ring system having one or more heteroatoms (i.e., non-carbon atoms). Preferred heterocycloalkyl groups include, for example, morpholino groups. As used herein, the term xe2x80x9cheterocycloalkenylxe2x80x9d denotes a ring system having one or more double bonds, and one or more heteroatoms. Preferred heterocycloalkenyl groups include, for example, pyrrolidino groups.
In some embodiments of the invention, A is a monocyclic or bicyclic aromatic ring system. Suitable monocyclic or bicyclic aromatic ring systems include phenyl, naphthyl, pyridyl, furyl and indolyl.
In some preferred embodiments of the compounds and methods of the invention, R11 and R12 can be, together with the carbon atoms to which they are attached, an optionally substituted aliphatic or aromatic ring having from 4 to 6 ring atoms. Examples of such rings include phenyl and naphthyl. Examples of substituents for such rings include halogen, hydroxyl, alkyl, and acetyl groups. In more preferred embodiments, R11 and R12 are each H.
In some preferred embodiments of the invention R2, or R3 can be a linker connected to a solid support. Solid supports are substrates which are capable of serving as the solid phase in solid phase synthetic methodologies, such as those described in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069. Linkers are known in the art as short molecules which serve to connect a solid support to functional groups (e.g., hydroxyl groups) of initial synthon molecules in solid phase synthetic techniques. Suitable linkers are disclosed in, for example, Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991, Chapter 1, pages 1-23.
Solid supports according to the invention include those generally known in the art to be suitable for use in solid phase methodologies, including, for example, controlled pore glass (CPG), oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527, hereby incorporated by reference in its entirety), TentaGel Supportxe2x80x94an aminopolyethyleneglycol derivatized support (see, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373, hereby incorporated by reference in its entirety) and Porosxe2x80x94a copolymer of polystyrene/divinylbenzene.
In some preferred embodiments of the invention R2, R3 or R3a can be a hydroxyl protecting group. A wide variety of hydroxyl protecting groups can be employed in the methods of the invention. Preferably, the protecting group is stable under basic conditions but can be removed under acidic conditions. In general, protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule. Representative hydroxyl protecting groups are disclosed by Beaucage, et al., Tetrahedron 1992, 48, 2223-2311, and also in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley and Sons, New York, 1991, each of which are hereby incorporated by reference in their entirety.
Preferred protecting groups used for R2, R3 and R3a include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl(Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl(Mox). The R2 or R3 group can be removed from oligomeric compounds of the invention by techniques well known in the art to form the free hydroxyl. For example, dimethoxytrityl protecting groups can be removed by protic acids such as formic acid, dichloroacetic acid, trichloroacetic acid, p-toluene sulphonic acid or with Lewis acids such as for example zinc bromide. See for example, Greene and Wuts, supra.
In some preferred embodiments of the invention amino groups are appended to alkyl or to other groups such as, for example, to 2xe2x80x2-alkoxy groups. Such amino groups are also commonly present in naturally occurring and non-naturally occurring nucleobases. It is generally preferred that these amino groups be in protected form during the synthesis of oligomeric compounds of the invention. Representative amino protecting groups suitable for these purposes are discussed in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 7, 2d ed, John Wiley and Sons, New York, 1991. Generally, as used herein, the term xe2x80x9cprotectedxe2x80x9d when used in connection with a molecular moiety such as xe2x80x9cnucleobasexe2x80x9d indicates that the molecular moiety contains one or more functionalities protected by protecting groups.
Sulfurizing agents used during oxidation to form phosphorothioate and phosphorodithioate linkages include Beaucage reagent (see e.g. Iyer, R. P., et.al., J. Chem. Soc., 1990, 112, 1253-1254, and Iyer, R. P., et.al., J. Org. Chem., 1990, 55, 4693-4699); tetraethylthiuram disulfide (see e.g., Vu, H., Hirschbein, B. L., Tetrahedron Lett., 1991, 32, 3005-3008); dibenzoyl tetrasulfide (see e.g., Rao, M. V., et.al., Tetrahedron Lett., 1992, 33, 4839-4842); di(phenylacetyl)disulfide (see e.g., Kamer, P. C. J., Tetrahedron Lett., 1989, 30, 6757-6760); Bis(O,O-diisopropoxy phosphinothioyl)disulfids (see Stec et al., Tetrahedron Lett., 1993, 34, 5317-5320); 3-ethoxy-1,2,4-dithiazoline-5-one (see Nucleic Acids Research, 1996 24, 1602-1607, and Nucleic Acids Research, 1996 24, 3643-3644); Bis(p-chlorobenzenesulfonyl)disulfide (see Nucleic Acids Research, 1995 23, 4029-4033); sulfur, sulfur in combination with ligands like triaryl, trialkyl, triaralkyl, or trialkaryl phosphines. The foregoing references are hereby incorporated by reference in their entirety.
Useful oxidizing agents used to form the phosphodiester or phosphorothioate linkages include iodine/tetrahydrofuran/ water/pyridine or hydrogen peroxide/water or tert-butyl hydroperoxide or any peracid like m-chloroperbenzoic acid. In the case of sulfurization the reaction-is performed under anhydrous conditions with the exclusion of air, in particular oxygen whereas in the case of oxidation the reaction can be performed under aqueous conditions.
Oligonucleotides or oligonucleotide analogs according to the present invention hybridizable to a specific target preferably comprise from about 5 to about 50 monomer subunits. It is more preferred that such compounds comprise from about 10 to about 30 monomer subunits, with 15 to 25 monomer subunits being particularly preferrred. When used as xe2x80x9cbuilding blocksxe2x80x9d in assembling larger oligomeric compounds (i.e., as synthons of Formula II), smaller oligomeric compounds are preferred. Libraries of dimeric, trimeric, or higher order compounds of general Formula II can be prepared for use as synthons in the methods of the invention. The use of small sequences synthesized via solution phase chemistries in automated synthesis of larger oligonucleotides enhances the coupling efficiency and the purity of the final oligonucloetides. See for example: Miura, K., et al., Chem. Pharm. Bull., 1987, 35, 833-836; Kumar, G., and Poonian, M. S., J. Org. Chem., 1984, 49, 4905-4912; Bannwarth, W., Helvetica Chimica Acta, 1985, 68, 1907-1913; Wolter, A., et al., nucleosides and nucleotides, 1986, 5, 65-77, each of which are hereby incorporated by reference in their entirety.
In one aspect of the invention, the compounds of the invention are used to modulate RNA or DNA, which code for a protein whose formation or activity it is desired to modulate. The targeting portion of the composition to be employed is, thus, selected to be complementary to the preselected portion of DNA or RNA, that is to be hybridizable to that portion.
In some preferred embodiments of the methods of the invention, compounds of Formula II are prepared by reaction of a protected nucleoside having Formula V: 
and a phosphine compound of Formula VI: 
in the presence of an acid. Suitable acids include those known in the art to be useful for coupling of phosphoramidites, including, for example, tetrazole, substituted tetrazoles, dicyanoimidazole, or diisopropylammonium tetrazolide.
In some preferreed embodiments, compounds of Formula VI are prepared by reacting an alcohol having the Formula XX: 
with phosphorus trichloride, and reacting the resultant product, Cl2Pxe2x80x94X1xe2x80x94(CH2)2xe2x80x94X5xe2x80x94C6H4xe2x80x94OX4, with at least two equivalents of an amine having the formula [(R6)2N]2NH. Each of the R6 groups can be the same or different, and are preferably alkyl having 1 to about 10 carbon atoms, more preferably 1 to 6 carbon atoms, with 3 carbon atoms, and particularly isopropyl groups, being especially preferred.
In further preferred embodiments, compounds of Formula II can be prepared by reaction of a protected nucleoside of Formula V with a chlorophosphine compund of formula ClP(R5)2, where R5 is preferably isopropylamino, followed by reaction with a compound of Formula XX in the presence of an acid, for example 1-H tetrazole, substituted tetrazoles, or dicyanoimidazole, with 1-H tetrazole being preferred.
In some particularly preferred embodiments of the foregoing methods, X4 is benzoyl, acetyl or levulinyl, A is phenyl with the moiety xe2x80x94OX4 being in the ortho or para position thereof, with the ortho position being more preferred, or A is naphthalene connected to X5 at the 1-position, with the moiety xe2x80x94OX4 being in the 5- or 6-position of the naphthalene ring.
In the compounds and methods of the present inventon, X1 and X2 can each independently be O or S. Thus, compounds having chiral phosphorus linkages are contemplated by the present invention. See Stec, W. J., and Lesnikowski, Z. J., in Methods in Molecular Biology Vol. 20: Protocols for Oligonucleotides and Analogs, S. Agrawal, Ed., Humana Press, Totowa, N.J. (1993), at Chapter 14. See also Stec, W. J. et al., Nucleic Acids Research, Vol. 19, No. 21, 5883-5888 (1991); and European Patent Application EP 0 506 242 A1, each of which are hereby incorporated by reference in their entirety.
Also provided in preferred embodiments of the invention are compounds having the general Formula VII: 
wherein X1, A, X4 and X5 and D are as defined above.
In particularly preferred embodiments, the compounds of the invention have the Formula II: 
wherein:
X4, M, X1, R1, X2, R3, B, n, and R5 are defined as above. In some especially preferred embodiments of the compounds of the invention having formula II above, X4 is benzoyl, acetyl or levulinyl, or a group of formula xe2x80x94(CH2xe2x80x94CH2)dSi (R9)3 where d is 0 or 1; A is phenyl having the moiety xe2x80x94OX4 is in the ortho or para position, with the ortho position being preferred, R5 is diisopropylamino, and n is 0.
The oligomeric compounds of the invention can be used in diagnostics, therapeutics and as research reagents and kits. They can be used in pharmaceutical compositions by including a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism should be contacted with an oligonucleotide having a sequence that is capable of specifically hybridizing with a strand of nucleic acid coding for the undesirable protein. Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms. Any organism that utilizes DNA-RNA transcription or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with the invention. Seemingly diverse organisms such as bacteria, yeast, protozoa, algae, all plants and all higher animal forms, including warm-blooded animals, can be treated. Further, each cell of multicellular eukaryotes can be treated, as they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity. Furthermore, many of the organelles (e.g., mitochondria and chloroplasts) of eukaryotic cells also include transcription and translation mechanisms. Thus, single cells, cellular populations or organelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic oligonucleotides.
As will be recognized, the steps of the methods of the present invention need not be performed any particular number of times or in any particular sequence. Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are intended to be illustrative and not intended to be limiting.